PROJECT SUMMARY/ABSTRACT: The diabetic wound healing impairment represents a major clinical problem, resulting in prolonged hospitalizations and significant healthcare expenditures. Two-thirds of non- traumatic amputations are preceded by a diabetic wound. The impaired healing of diabetic wounds has been shown to be multifactorial, however, increasing evidence suggests that persistent inflammation contributes to the pathogenesis of diabetic wounds through persistent activation of inflammatory pathways and increased oxidative stress. We have shown that diabetic wounds have increased expression of the proinflammatory cytokines IL-6 and IL-8, and decreased expression of the anti-inflammatory microRNA-146a, which inhibits NFkB activation and downstream IL-6 and IL-8 gene expression. In normal wound healing, macrophages are initially polarized to the proinflammatory M1 phenotype and then transition to the M2 phenotype, which is associated with resolution of inflammation and wound closure. Chronic inflammation and the associated ROS promote persistent proinflammatory M1 macrophage polarization and a failure to transition to the M2 phenotype, which has been implicated in the development of chronic diabetic wounds. We have designed novel cerium oxide nanoparticles (CNPs) that possess ROS scavenging properties and have conjugated them with a miR-146a mimetic, to synergistically target both proinflammatory signaling and ROS. In compelling preliminary data, we have found that one-time treatment of murine diabetic wounds with our novel miR-146a conjugated CNPs (CNP-miR146a) can improve diabetic wound healing, similar to that of non-diabetic wounds at 7 days, and this is associated with decreased inflammation and decreased expression of NOX2. In additional preliminary data, we have shown that CNP-miR146a can also improve wound healing in a streptozotocin porcine diabetes model. The objective of this work is to determine the mechanisms by which CNP-miR146a corrects the diabetic wound healing impairment, and validate this correction and toxicity in a preclinical porcine model. We hypothesize that CNP-miR146a will reduce inflammation and oxidative stress, thus driving macrophage transition from a proinflammatory (M1) to an anti-inflammatory (M2) phenotype and allow for resolution of the chronic inflammatory response and result in enhanced healing. Specific aim 1: To test whether CNP-miR146a corrects the diabetic wound healing impairment by decreasing inflammation and oxidative stress. Specific aim 2: To test whether decreased inflammation and oxidative stress with CNP-miR146a treatment improves healing by decreased proinflammatory (M1) and increased anti- inflammatory/resolving (M2) macrophage polarization. Specific aim 3: To validate that microRNA-146a conjugated CNPs correct the diabetic wound healing impairment and is non-toxic in a preclinical porcine model.